The present invention relates to immunological materials and methods useful for the determination of dihydropyrimidine dehydrogenase (DPD) in biological samples.
Dihydropyrimidine dehydrogenase (DPD) is an enzyme which catalyzes the reduction of pyrimidines to 5,6-dihydropyrimidines, where the resulting catabolites, dihydrouracil and dihydrothyrnine, are further catabolized to xcex2-alanine or xcex2-aminoisobutylic acid, respectively.
DPD is also known to catalyze the reducing reactions of 5-fluorouracil (5-FU) and various catabolites from pyrimidine analogues and derivatives, which are widely utilized as antitumor medicines, such as 2xe2x80x2-deoxy-5-fluorouridine (2xe2x80x2-dFUrd), 5xe2x80x2-deoxy-5-fluorouridine (5xe2x80x2-dFUrd, doxifluridine), and the like. It has been reported that the DPD level in biological samples of individuals suffering from tumor is an interesting marker of the therapeutic efficacy of antitumor medicaments in the series of 5-fluorouracil derivatives (see e. g. Iigo et al., 1969, Biochem. Pharmacol., 38, 1885-1889).
Deficiency in DPD is known to be responsible for the toxicity of the metabolites of pyrimidine antitumor medicines, which results in life-threatening condition during chemotherapy (Milano, G. and Etienne, 1988, M. C., Pharmacogenetics, 4, 301-306). Recently it was found that the disorder of DPD deficiency is more frequent than initially thought (Diasio and Lu, 1994, J. Clin. Oncol., 12, 2239-2242). It is thus important to identify patients with DPD deficiency.
There is therefore a need for a sensitive and reliable assay for the determination of the DPD level in biological samples.
Several methodologies for such assays have been described. Fernandez-Salguero et al. reported a thin layer chromatography (TLC) procedure for the determination of DPD activity in human peripheral lymphocytes (Fernandez-Salguero et al., 1995. Biochem. Pharm. 50, 1015-1020). The assay uses radiolabeled uracil as a substrate. However, a method which uses a radiolabel is obviously not adapted to routine diagnosis in hospitals. Furthermore, such a TLC method is time-consuming.
Other assay methods utilizing HPLC have been also described, e.g. by van Gennip et al., 1982, Adv. Exp. Med. Biol., 253A: 111-118, and Sommadossi et al., 1982, J. Biol. Chem., 257: 8171-8176. Those methods are based on the measurement of enzyme activity by the determination of the sum of various catabolites of 5-FU using reverse phase HPLC. Such methods are cumbersome and time-consuming.
The problem addressed by the present invention is to find materials and methods for the determination of DPD level that do not have the drawbacks of the above known methods, and that provide a simple and speedy assay for routine use in hospitals.
The above problem is solved by the invention as defined in the appended claims.
The present invention provides a monoclonal antibody specifically recognizing dihydropyrimidine dehydrogenase (DPD), and particularly human dihydropyrimidine dehydrogenase.
The above monoclonal antibody is suitably a monoclonal antibody that shows strong reactivity to a homogenate from human tumor cell line HT-3 (ATCC HTB-32), but a low reactivity or no reactivity to a homogenate from human tumor cell line MCF-7 (ATCC HTB-22).
The above monoclonal antibody is a monoclonal antibody that shows a high specificity for DPD.
Suitably the immunoprecipitate obtained with a homogenate from human tumor cell line HT-3 (ATCC HTB-32) will show a single band when analyzed by SDS-PAGE.
The monoclonal antibody can suitably be produced by a hybridoma cell line selected from the group consisting of hybridoma cell lines 2B6-11-1, 9C7-30-1, 5E6-19-1, 3A5-6-1 (FERM BP-6015), 6H5-42-1, 4B9-12-1 (FERM BP-6016), 2E2-B3-1-3 (FERM BP-6014) and 3B12-B1-56-1-2. Preferably the monoclonal antibody is one produced by a hybridoma cell line chosen among 3A5-6-1 (FERM BP-6015), 4B9-12-1 (FERM BP-6016), 2E2-B3-1-3 (FERM BP-6014).
The monoclonal antibody can also be any monoclonal antibody that binds to DPD in an equivalent manner as a monoclonal antibody produced by one of the above cell lines, i.e. a monoclonal antibody which binds to the same epitope as a monoclonal antibody produced by one of the above cell lines or crossreacts strongly with a monoclonal antibody produced by one of the above cell lines.
The invention also concerns a hybridoma cell line producing the above defined monoclonal antibody.
That hybridoma cell line is suitably chosen from the group consisting of hybridoma cell lines 2B6-11-1, 9C7-30-1, 5E6-19-1, 3A5-6-1 (FERM BP-6015), 6H5-42-1, 4B9-12-1 (FERM BP-6016), 2E2-B3-1-3 (FERM BP-6014) and 3B12-B1-56-1-2, and preferably a hybridoma cell line chosen among 3A5-6-1 (FERM BP-6015), 4B9-12-1 (FERM BP-6016), 2E2-B3-1-3 (FERM BP-6014).
The present invention also relates to a pair of monoclonal antibodies which is useful for the qualitative and/or quantitative detection of DPD, wherein the pair comprises a first monoclonal antibody specifically recognizing dihydropyrimidine dehydrogenase and a second monoclonal antibody specifically recognizing dihydropyrimidine dehydrogenase, wherein the first monoclonal antibody and the second monoclonal antibody recognize different epitopes from one another.
Such a pair of monoclonal antibodies can include as first monoclonal antibody, a monoclonal antibody selected from the group consisting of Mab-3A5-6-1, Mab-6 H5-42-1 and Mab-3B12-B1-56-1-2, and as second monoclonal antibody, a monoclonal antibody selected from the group consisting of Mab-4B9-12-1, Mab-2E2-B3-1-3, Mab-9C7-30-1, Mab-2B6-11-1 and Mab-5E6-19-1.
Preferably the first monoclonal antibody is Mab-3A5-6-1, and the second monoclonal antibody is Mab-4B9-12-1 or Mab-2E2-B3-1-3.
Further the present invention relates to kit for the detection and/or determination of the amount of dihydropyrimidine dehydrogenase in a biological sample which comprises
(a) at least one monoclonal antibody as defined above, and
(b) a label for qualitatively and/or quantitatively detecting the immunoconjugate of the monoclonal antibody and dihydropyrimidine dehydrogenase.
Preferably that kit comprises a pair of monoclonal antibodies and a label as defined above.
In another aspect, the invention relates to an immunoassay method for detecting and/or determining the amount of dihydropyrimidine dehydrogenase in a biological sample which comprises
(a) treating the sample with at least one monoclonal antibody as defined above, so to produce an immunoconjugate between that antibody and dihydropyrimidine dehydrogenase, and
(b) qualitatively or quantitatively detecting the said immunoconjugate with the aid of a label.
The immunoassay can be of any immunoassay format known in the art of immunoassays (see e.g. M. Ferencik, 1993, in xe2x80x9cHandbook of Immunochemistryxe2x80x9d, published by Chapman and Hall, London, UK). In particular it can be one-point binding assay, e.g. an immunoprecipitation method, or a two-point binding assay (sandwich assay), e.g. an ELISA. A two-point binding assay will preferably use a pair of monoclonal antibodies as defined above.
In a further aspect the invention relates to a method for the determination of the DPD deficiency state of a patient which comprises; (a) treating a biological sample from the patient with a monoclonal antibody as defined above, and (b) quantitatively detecting the immunoconjugate of the monoclonal antibody and dihydropyrimidine dehydrogenase.
In another aspect the invention concerns a method for estimating the susceptibility of patients suffering from cancer to the treatment with antitumor medicaments in the series of 5-fluorouracil (5-FU) derivatives, which comprises
(a) treating a biological sample from the patient with a monoclonal antibody as defined above,
(b) quantitatively detecting the immunoconjugate of the monoclonal antibody and human dihydropyrimidine dehydrogenase, so as to determine the level of dihydropyrimidine dehydrogenase in the biological sample from the patient to obtain a measure of susceptibility.
The above method enables the physician, by comparing that measured level of DPD for a patient to that of a reference set of patients, to predict the efficacy and toxicity of the above medicaments.
In further aspect the invention concerns a method for estimating the susceptibility of patients suffering from cancer to the treatment with antitumor medicaments in the series of 5-fluorouracil (5-FU) derivatives, which comprises
(a) treating a biological sample from the patient with a monoclonal antibody as defined above,
(b) quantitatively detecting the immunoconjugate of the monoclonal antibody human and dihydropyrimidine dehydrogenase, so as to determine the level of dihydropyrimidine dehydrogenase in the biological sample from the patient,
(c) determining the level of pyrimidine nucleoside phosphorylase in the said biological sample from the said patient, and
(d) calculating the ratio of the level of pyrimidine nucleoside phosphorylase and the level of dihydropyrimidine dehydrogenase to obtain a measure of susceptibility.
The above method enables the physician, by comparing that calculated ratio of the level of pyrimidine nucleoside phosphorylase and the level of dihydropyrimidine dehydrogenase for a patient and that of a reference set of patients, to predict the efficacy of the above medicaments.